Nucleic acids such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are used extensively in the field of molecular biology for research and clinical analyses. RNA may be found in nature in various forms, including messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), and viral RNA. Each of these types of RNA have distinct properties related to their specific functions. Analysis of RNA expression levels and patterns provides important information in fields such as developmental genetics, drug discovery and clinical diagnostics. For example, RNA analysis provides important diagnostic information about both normal and aberrant functioning of genes. Furthermore, gross DNA rearrangements associated with common leukemias are detected by isolation and identification of abnormal, hybrid RNAs.
Common methods for analyzing RNA include northern blotting, ribonuclease protection assays (RPAs), reverse transcriptase-polymerase chain reaction (RT-PCR), cDNA preparation for cloning, in vitro translation and microarray analyses. To obtain valid and consistent results from these analyses, it is important that the RNA be purified from other components common to biological materials such as proteins, carbohydrates, lipids and DNA.
RNA purification methods fall into two general categories, liquid phase and solid phase purification. In liquid phase purification, the RNA remains in the liquid phase while impurities are removed by processes such as precipitation and/or centrifugation. In solid phase purification, the RNA is bound to a solid support while impurities such as DNA, proteins, and phospholipids are selectively eluted. Both purification strategies utilize conventional methods, which require numerous steps and, often, hazardous reagents, as well as more rapid methods, which require fewer steps and usually less hazardous reagents. When the starting biological material comprises cells, both methods require a cell or viral co-rupture or lysis step that results in a mixed RNA with contaminants such as DNA, lipids, carbohydrates, proteins, etc. Such mixtures also contain RNases that easily degrade RNA and must be removed and/or inactivated.
Traditionally, liquid phase RNA isolation methods have used liquid-liquid extraction (i.e, phenol-chloroform) and alcohol precipitation. Perhaps the most commonly used liquid-liquid extraction method is the “acid-guanidinium-phenol” method of Chomczynski and Sacchi (Chomczynski P, Sacchi N., Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal Biochem 162: 156–9 [1987]; U.S. Pat. Nos. 5,945,515, 5,346,994, and 4,843,155). This method comprises: (1) extracting the sample with a guanidinium isothiocyanate (GITC) solution to which an acidic medium, phenol, and chloroform are added consecutively; (2) centrifuging the mixture to separate the phases such that the proteins denatured by the phenol may be removed from the nucleic acids that are found in an intermediate layer; (3) adding an alcohol so as to precipitate and thereby concentrate the RNA; and (4) washing and re-hydrating the purified RNA. Although this method ensures the purification of RNA, it utilizes hazardous reagents such as chloroform and phenol. Precipitation of nucleic acids by cationic detergents is another example of liquid phase technology (U.S. Pat. Nos. 5,985,572; 5,728,822 and 5,010,183 (MacFarlane)). For example, U.S. Pat. No. 5,985,572 discloses a novel method for isolating RNA from biological samples using selected quaternary amine surfactants. A non-hazardous liquid phase purification method was disclosed by Heath (U.S. Pat. No. 5,973,137) using low pH lysing and precipitation reagents. However, liquid phase methods have serious disadvantages in that they involve tedious precipitation steps, and are consequently difficult to automate. Thus, the need for high-throughput RNA purification has led to the development of solid phase methods.
As with liquid phase purification, conventional solid phase methods have been developed to generate highly purified RNA. Generally, these methods require four general steps: lysing cells or viral coats to release RNA; binding the released RNA to a solid support; washing away impurities; and then eluting the purified RNA. The first two steps, lysing the cells or viral coats and binding the released RNA, have traditionally required hazardous reagents.
For solid phase nucleic acid isolation methods, many solid supports have been used including membrane filters, magnetic beads, metal oxides, and latex particles. Probably the most widely used solid supports are silica-based particles (see, e.g., U.S. Pat. No. 5,234,809 (Boom et al.); International Publication No. WO 95/01359 (Colpan et al.); U.S. Pat. No. 5,405,951 (Woodard); International Publication No. WO 95/02049 (Jones); WO 92/07863 (Qiagen GmbH). One method for binding nucleic acids to silica is by the use of chaotropic agents. For example, the method disclosed in U.S. Pat. No. 5,234,809 (Boom et al.) uses a high concentration chaotropic solution such as guanidine isothiocyanate to bind DNA to silica particles and requires six centrifugation steps and five reagents to purify DNA from whole blood.
Specifically, Boom teaches (1) mixing the biological material with a solution consisting of guanidine isothiocyanate, EDTA and Triton X-100, and silica; (2) allowing the nucleic acid to bind to the silica; (3) washing the silica with consecutive washes of guanidine isothiocyanate, ethanol, acetone; and (4) eluting the nucleic acid with an eluent. Disadvantages of this method are the use of a particulate suspension, the use of many centrifugation steps, and the use of hazardous reagents, such as guanidine isothiocyanate and acetone. Furthermore, although this method has been employed successfully for DNA isolation, it is unsuitable for RNA isolation due to unacceptable levels of DNA contamination.
The use of chaotropic salts for the binding and purification of RNA is well known in the art. In another method See U.S. Pat. No. 5,990,302 (Kuroita et al.), the biological material is lysed in an acidic solution containing a lithium salt and a chaotropic agent such as guanidinium isothiocyanate (GITC), after which the RNA is brought into contact with a nucleic acid-binding carrier such as silica. The RNA is subsequently purified by eluting from the silica in a low ionic-strength buffer. This method is disadvantageous in its use of hazardous substances such as the chaotropic salt, guanidine isothiocyanate.
Combinations of chaotropic substances such as guanidine isothiocyanate, guanidine hydrochloride, sodium iodide, and urea mixtures at ionic strengths greater than 4 M in conjunction with silica-based carriers have been taught in the art. For example, Hillebrand et al. See WO 95/34569 describes a one-step method involving a slurry of silica beads to which chaotropic substances are added in order to cause RNA to bind.
The apparent opposite approach to the use of chaotropes is the use of antichaotropes (also known as “kosmotropes” in the art) to isolate DNA. See Hillebrand et al. US 20010041332. Hillebrand describes the use of “antichaotropes”, such as ammonium chloride (also cesium, sodium and/or potassium salts are mentioned), in combination with PVP (polyvinyl pyrrolidone) to lyse the starting sample and bind to the solid support with a detergent/alcohol mixture. Besides the fact that it is generally known that cesium and potassium are clearly considered to be chaotropes, due to their low charge density and weak hydration characteristics, while ammonium is considered to be a marginal chaotrope (Collins, K. Sticky Ions in Biological Systems, Proc. Natl. Acad. Sci. USA, 92 (1995), 5553–5557; Wiggins, P. M. High and Low Density Intracellular Water, Cellular and Molecular Biology 47 (5), 735–744), several disadvantages to the methods of Hillebrand exist. First, the methods use PVP, which has been investigated as a tumorigen. Secondly, heating steps of 65–70° C. are required for lysis and elution. Such heating may cause damage to the nucleic acids by nonspecific degradation or digestion resulting in limited downstream applications, such as incompatibility with restriction digests or blot analysis. Thirdly, the methods described by Hillebrand would not work effectively for isolating RNA because the methods do not employ reagents sufficient to reduce or eliminate RNases that are so troublesome in RNA purification.
Others have taught the use of ion-exchange resins to which nucleic acids bind at low pH and from which they are eluted at a higher pH. See U.S. Pat. No. 5,057,426 (Henco et al.). Such methods are primarily advantageous for the selective separation of long-chain nucleic acids which have a distinctive charge from smaller nucleic acids and other biological materials such as proteins. These methods are not successful for the isolation of RNA (irrespective of length and charge) from the remainder of the biological material. Furthermore, the long-chain nucleic acids must be eluted at high salt concentrations for an ion-exchange method to work. Commonly used salts (e.g., NaCl and KCl) can interfere with many enzymes used in molecular biology.
Polycationic solid supports have also been used in the purification of nucleic acids from solutions containing contaminants. See U.S. Pat. No. 5,599,667 (Arnold et al.). Polycationic supports selectively adsorb nucleotide multimers based on their size, the larger multimers having a higher affinity for the polycationic support than the smaller ones. This method is based largely on the affinity between positively charged cationic solid supports and negatively charged phosphate backbones of nucleotides. Larger nucleotide multimers have higher charges and will consequently bind preferentially over smaller nucleotide multimers. Thus, the method of Arnold is suited to the isolation of nucleotide multimers based on size rather than the isolation of all types of RNA from crude biological materials. Furthermore, the method of Arnold limits itself to the use of polycationic supports composed of cations such as ammonium, immonium and guanidinium ions.
A recent isolation method employs the principle that RNA precipitates preferentially in the presence of guanidinium salts under defined buffer conditions. See U.S. Pat. No. 5,972,613 (Somack et al.). In this method, RNA is precipitated in the presence of guanidinium salts at low temperatures, while the DNA remains in solution. A commonly used method for precipitation of RNA from solution involves the use of LiCl salt. In one version of this method, 0.8 M LiCl is used to selectively precipitate large ribosomal and messenger RNA molecules from a solution containing total RNA molecules (Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) in Molecular Cloning A Laboratory Manual, Vol. 3, 2nd ed., Appendices E.10 & E.15). In a another version of this method, 2.5 M LiCl is used to selectively precipitate RNA molecules as small as 100 nucleotides in length, from solutions containing total RNA and single nucleotides (Ambion technical bulletin #160).
The method of selectively precipitating RNA from a solution containing nucleic acids and other biological materials is physically different than that of using a solid phase to selectively bind the RNA molecules in a solution. A precipitation event is the reverse of a solution event. Solution involves the dissolving of a solute, such as RNA, by separation of that solute into molecules that are surrounded by solvent. Precipitation involves the removal of solvent and coalescence of individual RNA molecules into a solid which separates from the solvent. These precipitation and solution events occur within a solution environment and do not depend upon a separate and distinct solid phase on which RNA purification and separation occurs.
To further advance the field of RNA purification there is a need for solid phase RNA purification strategies. There is also a need for reagents and methods that are adaptable to solid phase purification strategies which are not only simple and rapid, but general in scope to maximize adaptability for automation. There is a need for reagents that are stable at room temperature (i.e., 20–25° C.), less hazardous (i.e., less corrosive or toxic), nonparticulate to eliminate the need for mixing, and protective of RNA quality. There is also a need for methods with few steps that can be performed using a variety of biological starting materials, whether hydrated or dried, especially as applied to routine testing as found in clinical and research laboratories. In addition the RNA purification reagents must not inhibit subsequent RNA analysis procedures by carrying over particulates or interfering with the buffering capacity or ionic conditions of downstream analyses such as: reverse transcriptase reactions, amplification reactions, nuclease protection assays, northern blotting, and microarray and other labeling reactions.